Methods for treating and/or limiting development of diabetes

ABSTRACT

The present invention provides methods for identifying candidate compounds for limiting development of and/or treating diabetes, and methods for limiting development of and/or treating diabetes.

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No. 14/717,903filed May 20, 2015, which is a divisional of Ser. No. 14/026,145 filedSep. 13, 2013, which is a continuation of U.S. application Ser. No.13/532,601 filed Jun. 25, 2012, now U.S. Pat. No. 8,557,513 issued onOct. 15, 2013, which claims priority to U.S. Provisional PatentApplication Ser. No. 61/501,480 filed Jun. 27, 2011, each incorporatedby reference herein in its entirety.

INTRODUCTION

Voltage-gated calcium (Ca_(v)) channels are critical in β cellphysiology and pathophysiology. They are not only take center stage inthe regulation of insulin secretion, but are also involved in β celldevelopment, survival and growth through the regulation of proteinphosphorylation, gene expression and the cell cycle. The function anddensity of β cell Ca_(v) channels are regulated by a wide range ofmechanisms either shared by other cell types or specific to β cells,e.g., channel phosphorylation, interaction with other molecules andglucose metabolism-derived signaling. Dysfunctional Ca_(v) channelscauses β cell malfunction and even death as manifested in the mostcommon metabolic disorder diabetes mellitus. Indeed, aT-lymphocyte-mediated autoimmune attack plays a crucial role in β celldeath in type 1 diabetes. In addition, factors in type 1 diabetic serumcompel unphysiological amounts of Ca²⁺ to enter pancreatic β cellsthrough hyperactivation of β cell Ca_(v) channels resulting in β cellapoptosis. Undoubtedly, this process aggravates the disease developmenton top of the autoimmune attack. Such factors are also visualized intype 2 diabetic serum where they behave in the same way as they do intype 1 diabetic serum. In fact, reduction in β cell mass andhyperactivation of β cell Ca_(v) channels appear under type 2 diabeticconditions such as those in the Goto-Kakizaki rat.

It has been demonstrated that elevated apolipoprotein CIII (ApoCIII)acts as a diabetogenic serum factor to drive β cell destruction viahyperactivation of β cell Ca_(v) channels. Moreover, we have recentlyshown that in vivo suppression of ApoCIII delays onset of diabetes inthe BioBreeding™ rat. Normally, ApoCIII is a blood plasma component. Itis synthesized predominantly in the liver and to a minor extent in theintestine. Liver and intestinal cells release this apolipoprotein intothe blood where it is situated on the surface of chylomicrons, very lowdensity lipoproteins (LDLs) and high density lipoproteins (HDLs).ApoCIII is composed of 79 amino acid residues that form six amphiphilicα-helixes, each containing about 10 residues. The three-dimensional NMRstructure and dynamics of ApoCIII have been resolved when it complexeswith sodium dodecyl sulfate micelles, mimicking its natural lipid-boundstate. The six amphiphilic α-helixes assemble into a necklace-like chainwrapping around the sodium dodecyl sulfate micelle surface.Dogmatically, ApoCIII serves as an effective inhibitor of triglyceridehydrolysis by inhibiting lipoprotein lipase and through interferencewith triglyceride-rich lipoproteins binding to the negatively chargedcell surface where lipoprotein lipases and lipoprotein receptors reside.It impedes the selective uptake of cholesteryl esters from LDL and HDLby binding to the scavenger receptor class B type I (SR-BI), and hampersthe endocytosis of cholesterol-rich LDL by prevention of apolipoproteinB binding to receptors. Elevated plasma ApoCIII concentration is afeature of dyslipidemia in obesity and observed in both type 1 and type2 diabetes, whereas a group of Ashkennazi Jewish with reduced plasmaApoCIII concentration maintains cardiovascular health and greaterinsulin sensitivity with age and reaches exceptional longevity.

In addition to the dogmatic roles in lipid metabolism, ApoCIII is also amultifaceted player in cell signaling. It can bind to distinct cellsurface receptors including scavenger receptor class B type I (SR-BI),Toll-like receptor 2 (TLR2) and uncharacterized binding sites relayingcorresponding signals to their downstream effectors, e.g., β1 integrin,pertussis toxin-sensitive G proteins, NF-κB and protein kinases.However, nothing is known about molecular mechanisms whereby ApoCIIIhyperactivates β cell Ca_(v) channels.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides methods identifyingcandidate compounds for limiting development of and/or treatingdiabetes, comprising

-   -   a) contacting a first population of insulin secreting cells with        an amount of apolipoprotein CIII (ApoCIII) effective to increase        density and/or conductivity of Ca_(v)1 channels, in the presence        of one or more test compounds; and    -   b) identifying those positive test compounds that inhibit an        ApoCIII-induced increase in density and/or conductivity of        Ca_(v)1 channels in the first population of insulin secreting        cells compared to control, wherein the positive test compounds        are candidate compounds for limiting development of and/or        treating diabetes.

In one embodiment, the control comprises contacting a control populationof insulin secreting cells with an amount of apolipoprotein CII(ApoCIII) effective to increase density and/or conductivity of Ca_(v)1channels, in the absence of one or more test compounds. This embodiment,which can be combined with the use of any of the other controlsdisclosed herein unless the context clearly dictates otherwise, maycomprise, for example, contacting the control population of cells with aformulation, such as a buffer, that is similar to or identical to theformulation the test compounds are dissolved in.

In another embodiment, the control comprises contacting a controlpopulation of insulin secreting cells with an amount of ApoCIIIeffective to increase density and/or conductivity of Ca_(v)1 channels,in the presence of the one or more test compounds, and furthercontacting the control population of insulin secreting cells withCa_(v)2 and/or Ca_(v)3 channel blockers, wherein positive test compoundsthat inhibit the ApoCIII-induced increase in density and/or conductivityof Ca_(v)1 channels in the Ca_(v)2 and/or Ca_(v)3 channel blockercontrol population of insulin secreting cells to a greater degree thanin the first population of insulin secreting cells are candidatecompounds for limiting development of and/or treating diabetes.

In another embodiment, the control comprises contacting a controlpopulation of insulin secreting cells with an amount of ApoCIIIeffective to increase density and/or conductivity of Ca_(v)1 channels,in the presence of the one or more test compounds, and furthercontacting the second population of insulin secreting cells with a Srckinase inhibitor and/or a protein kinase A (PKA) inhibitor, whereinpositive test compounds that inhibit the ApoCIII-induced increase indensity and/or conductivity of Ca_(v)1 channels in the first populationof insulin secreting cells to a greater degree than in the Src kinaseinhibitor and/or a protein kinase A (PKA) inhibitor control populationof insulin secreting cells are candidate compounds for limitingdevelopment of and/or treating diabetes.

In a further embodiment, the control comprises contacting a controlpopulation of insulin secreting cells with an amount of ApoCIIIeffective to increase density and/or conductivity of Ca_(v)1 channels,in the presence of the one or more test compounds, and furthercontacting the control population of insulin secreting cells with amolecule that inhibits β1 integrin expression or activity, whereinpositive test compounds that inhibit the ApoCIII-induced increase indensity and/or conductivity of Ca_(v)1 channels in the first populationof insulin secreting cells to a greater degree than in the β1 integrinexpression or activity control population of insulin secreting cells arecandidate compounds for limiting development of and/or treatingdiabetes.

As will be understood by those of skill in the art, a single control canbe used in carrying out the methods of the invention, including but notlimited to any of the controls disclosed above. Alternatively, multiplecontrol embodiments can be used (2, 3, or more, including but notlimited to any of the controls disclosed above), wherein each embodimentutilizes a different control cell population.

In a second aspect, the present invention provides methods foridentifying candidate compounds for limiting development of and/ortreating diabetes, comprising

-   -   (a) contacting a first population of insulin secreting cells        with an amount of ApoCIII effective to increase density and/or        conductivity of Ca_(v)1 channels, in the presence of one or more        test compounds; and    -   (b) identifying those positive test compounds that inhibit β1        integrin expression or activity in the first population of        insulin secreting cells compared to control, wherein the        positive test compounds are candidate compounds for limiting        development of and/or treating diabetes.

In one embodiment, the control comprises contacting a second populationof insulin secreting cells contacted with an amount of ApoCIII effectiveto increase density and/or conductivity of Ca_(v)1 channels, in theabsence of one or more test compounds. This embodiment may comprise, forexample, contacting the second population of cells with a formulation,such as a buffer, that is similar to or identical to the formulation thetest compounds are dissolved in.

In a third aspect, the present invention provides methods foridentifying candidate compounds for limiting development of and/ortreating diabetes, comprising

-   -   (a) contacting a first population of insulin secreting cells        with an amount of ApoCIII effective to increase density and/or        conductivity of Ca_(v)1 channels, in the presence of one or more        test compounds; and    -   (b) identifying those positive test compounds that inhibit        activation of PKA and/or Src kinase in the first population of        insulin secreting cells compared to control, wherein the        positive test compounds are candidate compounds for limiting        development of and/or treating diabetes.

In one embodiment, the control comprises contacting a second populationof insulin secreting cells contacted with an amount of ApoCIII effectiveto increase density and/or conductivity of Ca_(v)1 channels, in theabsence of one or more test compounds. This embodiment may comprise, forexample, contacting the second population of cells with a formulation,such as a buffer, that is similar to or identical to the formulation thetest compounds are dissolved in.

In various embodiments of any of these aspects of the invention, each ofwhich can be combined except as clearly dictated otherwise by thecontext, the method comprises contacting the cells with ApoCIII for atleast 6 hours; the candidate compounds are candidate compounds forlimiting development of and/or treating type 1 diabetes; and/or whereinthe candidate compounds are candidate compounds for limiting developmentof and/or treating type 2 diabetes.

In a fourth aspect, the present invention provides methods for treatingor limiting development of diabetes, comprising administering to asubject in need thereof with an amount effective of an inhibitor of PKA.and Src kinase to treat or limit development of diabetes.

In a fifth aspect, the present invention provides methods for treatingor limiting development of diabetes, comprising administering to asubject in need thereof with an amount effective of an inhibitor of β1integrin expression and/or activity. In various embodiments, theinhibitor is selected from the group consisting of an anti-β1 integrinantibody, anti-β1 integrin aptamer, β1 integrin siRNA, β1 integrinshRNA, and β1 integrin antisense oligonucleotides.

In a sixth aspect, the present invention provides methods for treatingor limiting development of diabetes, comprising administering to asubject in need thereof with an amount effective of an inhibitor ofApoCIII binding to pancreatic β cells. In various embodiments, theinhibitor is selected from the group consisting of aptamers andantibodies selective for ApoCIII.

DESCRIPTION OF THE FIGURES

FIG. 1. Apolipoprotein CIII incubation increases both the density andconductivity of Ca_(v)1 channels in β cells, (A) Examples of unitaryCa_(v)1 channel currents detected in plasma membrane patches of mouseislet β cells incubated with either vehicle solution as control orapolipoprotein CIII (ApoCIII). (B) Average number, open probability,mean closed time and mean open time of unitary Ca_(v)1 channels measuredin plasma membrane patches attached to mouse islet β cells exposed toeither control vehicle (n=33) or ApoCIII (n=32). (C) Examples of unitaryCa_(v)1 channel currents recorded in plasma membrane patches attached toeither a control RINm5F cell or a cell treated with ApoCIII. (D) Averagenumber, open probability, mean closed time and mean open time of unitaryCa_(v)1 channels detected in plasma membrane patches of control RINm5Fcells (n=34) or cells incubated with ApoCIII (n=35). *P<0.05 and**P<0.01 versus control.

FIG. 2. Apolipoprotein CIII incubation increases whole-cell Ca²⁺currents and coincubation with the Ca_(v)1 channel blocker nimodipineabrogates the effect of apolipoprotein CIII incubation in RINm5F cells.(A) Sample whole-cell Ca²⁺ current traces from a cell incubated withvehicle solution as control (cell capacitance: 10.1 pF) andapolipoprotein CIII (ApoCIII)-treated cell (cell capacitance: 11.1 pF).(B) Average Ca²⁺ current density-voltage relationships in control cells(open circles, n=26) and cells treated with ApoCIII (filled circles,n=26). *P<0.05 and **P<0.01 versus control. (C) Sample whole-cell Ca²⁺current traces from a nimodipine (Nim)-incubated cell (cell capacitance:10 pF) and a cell exposed to Nim together with ApoCIII (Nim/ApoCIII)(cell capacitance: 11.9 pF). (D) Average Ca²⁺ current density-voltagerelationships in Nim-treated cells (open circles, n=20) and cellsincubated with Nim/ApoCIII (filled circles, n=21). *P<0.05 and **P<0.01versus Nim alone.

FIG. 3. PKA or Src kinase inhibition marginally reduces, but PKCinhibition does not affect apolipoprotein CIII-induced enhancement ofwhole-cell Ca²⁺ currents in RINm5F cells. (A) Sample whole-cell Ca²⁺current traces from a cell incubated with vehicle solution as control(cell capacitance: 8.5 pF), an apolipoprotein CIII (ApoCIII)-treatedcell (cell capacitance: 8.2 pF) and a cell exposed to ApoCIII plus thePKA inhibitor H-89 (ApoCIII/H-89, cell capacitance: 8.4 pF). (B) AverageCa²⁺ current density-voltage relationships in control cells (opencircles, n=37) and cells treated with ApoCIII (filled circles, n36) orApoCIII/H-89 (filled triangles, n36), *P<0.05 and **P<0.01 versuscontrol. (C) Sample whole-cell Ca²⁺ current traces registered in acontrol cell (cell capacitance: 12.5 pF), ApoCIII-incubated cell (cellcapacitance: 12.0 pF) and a cell subjected to cotreatment with ApoCIIIand the PKC inhibitor calphostin C (ApoCIII/CalpC, cell capacitance:12.1 pF). (D) Average Ca²⁺ current density-voltage relationships incontrol cells (open circles, n33), ApoCIII-treated cells (filledcircles, n33) and cells exposed to ApoCIII/CalpC (filled triangles,n=33). *P<0.05 and **P<0.01 ApoCIII versus control. ⁺P<0.05 and ⁺⁺P<0.01ApoCIII/CalpC versus control. (E) Sample whole-cell Ca²⁺ current tracesacquired in a control cell (cell capacitance: 9.5 pF), anApoCIII-incubated cell (cell capacitance: 9.2 pF) and a cell exposed toApoCIII together with the Src kinase inhibitor PP2 (ApoCIII/PP2, cellcapacitance: 10.0 pF). (F) Average Ca²⁺ current density-voltagerelationships in control cells (open circles, n=40) and cells incubatedwith ApoCIII (filled circles, n=40) or ApoCIII/PP2 (filled triangles,n=40). **P<0.01 ApoCIII versus control. ⁺P<0.05 ApoCIII/PP2 versuscontrol.

FIG. 4. Combined inhibition of PKA, PKC and Src kinase counteractsapolipoprotein CIII-induced augmentation of whole-cell Ca²⁺ currents inRINm5F cells and coinhibition of PKA and Src kinase is sufficient toobtain this counteraction. (A) Sample whole-cell Ca²⁺ current tracesregistered in a vehicle-incubated cell (Control, cell capacitance: 7.9pF), a cell subsequent to apolipoprotein (ApoCIII) treatment (cellcapacitance: 7.0 pF) and a cell exposed to ApoCIII in the presence ofthe protein kinase inhibitor cocktail of H-89, calphostin C and PP2(ApoCIII/H-89/CalpC/PP2, cell capacitance: 7.2 pF). (B) Average Ca²⁺current density-voltage relationships in control cells (n=35) and cellsexposed to ApoCIII (n=34) or to ApoCIII/H-89/CalpC/PP2 (n=35). *P<0.05versus control and apoCIII/H-89/CalpC/PP2. (C) Sample whole-cell Ca²⁺current traces from a control cell (cell capacitance: 8.5 pF), a cellsubsequent to ApoCIII treatment (cell capacitance: 8.2 pF) and a cellexposed to ApoCIII in the presence of the protein kinase inhibitors H-89and PP2 (ApoCIII/H-89/PP2, cell capacitance: 8.7 pF). (D) Average Ca²⁺current density-voltage relationships in control cells (n=26) and cellssubjected to ApoCIII (n=26) or to ApoCIII/H-89/PP2 (n=27). *P<0.05 and**P<0.01 versus control; ⁺P<0.05 versus ApoCIII/H-89/PP2.

FIG. 5. Apolipoprotein CIII incubation does not alter β cell Ca_(v)1channel expression. (A) Representative immunoblots of RINm5F cellhomogenates, subjected to incubation with vehicle as control orapolipoprotein (ApoCIII), probed with anti-Ca_(v)1.2, anti-Ca_(v)1.3 andanti-GAPDH antibodies, respectively. (B) Immunoblot quantification ofthe relative abundance of Ca_(v)1.2 (hatched column, n=6) and Ca_(v)1.3subunits (filled column, n=6) in RINm5F cell homogenates subjected toApoCIII incubation in comparison with control (open column, n=6). Therewas no significant difference in the relative abundance of totalCa_(v)1.2 and Ca_(v)1.3 subunits between control cells and cellsincubated with ApoCIII (P>0.05).

FIG. 6. Knockdown of β1 integrin abrogates apolipoprotein CIII-inducedenhancement of whole-cell Ca²⁺ currents in RINm5F cells. (A)Representative blots of β1 integrin- and GAPDH-immunoreactive bands inβ1 integrin siRNA #1-, negative control siRNA (NC siRNA)- and β1integrin siRNA #2-transfected cells. (B) Immunoblot quantifications ofα1 integrin protein in NC siRNA—(open column, n=6), β1 integrin siRNA#1—(hatched column, n=6) and β1 integrin siRNA #2-transfected RINm5Fcells (filled column, n=6). **P<0.01 versus NC siRNA. (C) Samplewhole-cell Ca²⁺ current traces registered in individual cells followingmock transfection and incubation with control vehicle (NO siRNA/Control,cell capacitance: 12.1 pF), NC siRNA transfection and control vehicletreatment (NC siRNA/Control, cell capacitance: 11.4 pF), NC siRNAtransfection and apolipoprotein CIII (ApoCIII) incubation (NCsiRNA/ApoCIII, cell capacitance: 12.1 pF), β1 integrin siRNAtransfection and exposure to vehicle solution (β1 integrinsiRNA/Control, cell capacitance: 11.9 pF) and β1 integrin siRNAtransfection and ApoCIII exposure (β1 integrin siRNA/ApoCIII, cellcapacitance: 12.4 pF), respectively. (D) Ca²⁺ current density-voltagerelationships in cells subjected to NO siRNA/Control (filled circles,n=29), NC siRNA/Control (open circles, n=28), NC siRNA/apoCIII (filledtriangles, n=28), β1 integrin siRNA/Control (open triangles, n=29) andβ1 integrin siRNA/ApoCIII (filled squares, n=29). *P<0.05 and **P<0.01versus NO siRNA/Control, NC siRNA/Control and β1 integrin siRNA/Control.⁺P<005 versus β1 integrin siRNA/ApoCIII.

FIG. 7. PKA., PKC or Src kinase inhibition does not alter whole-cellCa²⁺ currents in RINm5F cells under basal conditions. (A) Samplewhole-cell Ca⁺ current traces from a vehicle-treated cell as control(cell capacitance: 8.8 pF) and a cell exposed to H-89 (cell capacitance:8.5 pF). (B) Average Ca²⁺ current density-voltage relationships incontrol cells (open circles; n=20) and cells incubated with H-89 (filledcircles, n=20). (C) Sample whole-cell Ca²⁺ current traces recorded in acontrol cell (cell capacitance: 10.4 pF) and a cell subjected tocalphostin C incubation (CalpC, cell capacitance: 11.0 pF). (D) AverageCa²⁺ current density-voltage relationships in control cells (opencircles; n=29) and cells exposed to CalpC (filled circles, n=29). (E)Sample whole-cell Ca²⁺ current traces obtained in a control cell (cellcapacitance: 9.0 pF) and a PP2-treated cell (cell capacitance: 9.1 pF).(F) Average Ca²⁺ current density-voltage relationships in control cells(open circles, n=20) and cells incubated with PP2 (filled circles,n=19).

FIG. 8. Combined inhibition of PKA, PKC and Src kinase or coinhibitionof PKA and Src kinase does not influence whole-cell Ca²⁺ currents inRINm5F cells under basal conditions. (A) Sample whole-cell Ca²⁺ currenttraces obtained in a cell incubated with vehicle solution as control(cell capacitance: 10.8 pF) and a cell treated with the protein kinaseinhibitor cocktail composed of H-89, calphostin C and PP2(H-89/CalpC/PP2, cell capacitance: 9.7 pF). (B) Average Ca²⁺ currentdensity-voltage relationships in control cells (open circles, n=30) andcells treated with H-89/CalpC/PP2 (filled circles, n=30). (C) Samplewhole-cell Ca²⁺ current traces obtained in a vehicle-treated cell ascontrol (cell capacitance: 9.4 pF) and a cell treated with the proteinkinase inhibitors H-89 and PP2 (H-89/PP2, cell capacitance: 9.1 pF). (D)Average Ca²⁺ current density-voltage relationships in control cells(open circles, n=24) and cells treated with H-89/PP2 (filled circles,n=24).

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides methods identifyingcandidate compounds for limiting development of and/or treatingdiabetes, comprising

-   -   (a) contacting a first population of insulin secreting cells        with an amount of apolipoprotein CIII (ApoCIII) effective to        increase density and/or conductivity of Ca_(v)1 channels, in the        presence of one or more test compounds; and    -   (b) identifying those positive test compounds that inhibit an        ApoCIII-induced increase in density and/or conductivity of        Ca_(v)1 channels in the first population of insulin secreting        cells compared to control, wherein the positive test compounds        are candidate compounds for limiting development of and/or        treating diabetes.

The inventors have discovered that ApoCIII incubation caused significantincreases in Ca_(v)1 channel open probability and density at singlechannel levels. The treatment significantly enhanced whole-cell Ca²⁺currents and the Ca_(v)1 channel blocker nimodipine completely abrogatedthe enhancement. The inventors have further discovered that coinhibitionof PKA and Src kinase was sufficient for the same counteraction, andthat knockdown of β1 integrin prevented ApoCIII from hyperactivating βcell Ca_(v) channels. Thus, the inventors have pinpointed therapeutictargets for limiting Ca²⁺-dependent pancreatic β cell death, and thusfor limiting and/or treating diabetes mellitus. Thus, the methods ofthis aspect of the invention can be used to identify compounds forlimiting Ca²⁺-dependent pancreatic β cell death and thus for limitingdevelopment of and/or treating diabetes.

As used herein, “apoCIII” refers to a protein comprising the amino acidsequence shown in SEQ ID NO:2 (Human) (NCBI accession number CAA25233),SEQ ID NO:4 (Rat) (NCBI accession number AA40746), or SEQ NO:6 (Macaque)(NCBI accession number CAA48419), or functional equivalents thereof.

The apoCIII may be substantially purified apoCIII, available, forexample, from Sigma Chemical Company (St. Louis, Mo.), wherein“substantially purified” means that it is removed from its normal invivo cellular environment. Alternatively, the apoCIII may be present ina mixture, such as blood serum from type I diabetic or partially orfully purified therefrom using standard techniques, such as thosedescribed below. In a preferred embodiment, substantially purifiedapoCIII is used.

As discussed below, there are three known isoforms of human apoCIII thathave the same amino acid sequence, but which differ in theirglycosylation pattern. Thus, in a preferred embodiment, glycosylatedapoCIII is used, wherein the glycosylation is preferably sialylation. Inanother preferred embodiment, mono-sialylated or di-sialylated apoCIIIis used. Such glycosylated forms may be purchased, for example, fromSigma Chemical Company, or may be partially or fully purified usingstandard techniques, such as those described below.

Any suitable insulin secreting cell can be used, including but notlimited to pancreatic β cells. As used herein, “pancreatic β cells” areany population of cells that contain pancreatic β islet cells. The cellscan be obtained from any mammalian species, or may be present within themammalian species when the assays are conducted in vivo. Such pancreaticβ islet cell populations include the pancreas, isolated pancreaticislets of Langerhans (“pancreatic islets”), isolated pancreatic β isletcells, and insulin secreting cell lines. Methods for pancreaticisolation are well known in the art, and methods for isolatingpancreatic islets, can be found, for example, in Cejvan et al., Diabetes52:1176-1181 (2003); Zambre et al., Biochem. Pharmacol. 57:1159-1164(1999), and Fagan et al., Surgery 124:254-259 (1998), and referencescited therein. Insulin secreting cell lines are available from theAmerican Tissue Culture Collection (“ATCC”) (Rockville, Md.). In afurther embodiment where pancreatic β cells are used, they are obtainedfrom ob/ob mice, which contain more than 95% β cells in their islets,and are commercially available.

Measuring the density and/or conductivity of Ca_(v)1 channels can becarried out by standard methods in the art, including but not limited tosingle channel and whole-cell patch-clamp measurements (cell-attachedand perforated whole-cell patch-clamp techniques). As used herein,“increase density and/or conductivity of Ca_(v)1 channels” refers toincreasing during the course of the assay above that seen in the absenceof test compounds. The method does not require a specific amount ofincrease in density and/or conductivity of Ca_(v)1 channels overbaseline, so long as the compound(s) promotes an increase in densityand/or conductivity of Ca_(v)1 channels above that seen in the absenceof test compounds. In a preferred embodiment, the increase is astatistically significant increase as judged by standard statisticalanalysis.

The contacting of the pancreatic β cells with the apoCIII may occurbefore, after, or simultaneously with contacting the cells with one ormore test compounds. The contacting can be in vitro or in vivo (ex: inan experimental animal model). Any suitable culture conditions can beused for carrying out the methods of any of the candidate identificationmethods of the invention. In one embodiment, the cells are contactedwith ApoCIII for at least 6 hours. In another embodiment, the cells aregrown in medium comprising between 1 mM and 15 mM glucose; preferablybetween 3 mM and 12 mM; preferably about 11 mM glucose. In a furtherembodiment, the cells are cultured at approximately 37° C. (preferablyin a humidified incubator, such as 5% CO₂) prior to recording thedensity and/or conductivity of the Ca_(v)1 channels at approximatelyroom temperature. These and other suitable assay conditions are wellwithin the level of those of skill in the art, based on the teachingsherein.

In one embodiment, the candidate compounds are candidate compounds forlimiting development of and/or treating type 1 diabetes. In anotherembodiment, the candidate compounds are candidate compounds for limitingdevelopment of and/or treating type 2 diabetes. The present inventionfurther provides compounds identified by the above screening methods,and their use for treating subjects in need thereof.

In another embodiment, the methods further comprise large-scalesynthesis of the test compounds that inhibit apoCIII-induced increase indensity and/or conductivity of Ca_(v)1 channels in the pancreatic βcells.

When the test compounds comprise polypeptide sequences, suchpolypeptides may be chemically synthesized or recombinantly expressed.Recombinant expression can be accomplished using standard methods in theart, as disclosed above. Such expression vectors can comprise bacterialor viral expression vectors, and such host cells can be prokaryotic oreukaryotic. Synthetic polypeptides, prepared using the well-knowntechniques of solid phase, liquid phase, or peptide condensationtechniques, or any combination thereof, can include natural andunnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl(Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Nα-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, such as by using automated synthesizers.

When the test compounds comprise antibodies, such antibodies can bepolyclonal or monoclonal. The antibodies can be humanized, fully human,or murine forms of the antibodies. Such antibodies can be made bywell-known methods, such as described in Harlow and Lane, Antibodies; ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1988).

When the test compounds comprise nucleic acid sequences, such nucleicacids may be chemically synthesized or recombinantly expressed as well.Recombinant expression techniques are well known to those in the art(See, for example, Sambrook, et al., 1989, supra). The nucleic acids maybe DNA or RNA, and may be single stranded or double. Similarly, suchnucleic acids can be chemically or enzymatically synthesized by manualor automated reactions, using standard techniques in the art. Ifsynthesized chemically or by in vitro enzymatic synthesis, the nucleicacid may be purified prior to introduction into the cell. For example,the nucleic acids can be purified from a mixture by extraction with asolvent or resin, precipitation, electrophoresis, chromatography, or acombination thereof. Alternatively, the nucleic acids may be used withno or a minimum of purification to avoid losses due to sampleprocessing.

When the test compounds comprise compounds other than polypeptides,antibodies, or nucleic acids, such compounds can be made by any of thevariety of methods in the art for conducting organic chemical synthesis.

In one embodiment, the control comprises contacting a second populationof insulin secreting cells with an amount of apolipoprotein CIII(ApoCIII effective to increase density and/or conductivity of Ca_(v)1channels, in the absence of one or more test compounds. This embodiment,which can be combined with the use of any of the other controlsdisclosed herein, may comprise, for example, contacting the secondpopulation of cells with a formulation, such as a buffer, that issimilar to or identical to the formulation the test compounds aredissolved in.

In one embodiment, the control comprises contacting a control populationof insulin secreting cells with an amount of ApoCIII effective toincrease density and/or conductivity of Ca_(v)1 channels, in thepresence of the one or more test compounds, and further contacting thecontrol population of insulin secreting cells with Ca_(v)2 and/orCa_(v)3 channel blockers, including hut not limited to ω-agatoxin IVA,ω-conotoxin GVIA and SNX 482 (Ca_(v)2 channel blockers); and mibefradiland NNC 55-0396 (Ca_(v)3 channel blockers), wherein positive testcompounds that inhibit the ApoCIII-induced increase in density and/orconductivity of Ca_(v)1 channels in the Ca_(v)2 and/or Ca_(v)3 channelblocker-control population of insulin secreting cells to a greaterdegree than in the first population of insulin secreting cells arecandidate compounds for limiting development of and/or treatingdiabetes. In this embodiment, the Ca_(v)2 and/or Ca_(v)3 channel blockerare selective for the Ca_(v)2 and/or Ca_(v)3 channel, and do not serveas a Ca_(v)1 channel blocker. It is within the level of those of skillin the art to determine, based on the teachings herein, the amount ofany Ca_(v)2 and/or Ca_(v)3 channel blocker(s) that can be usefully usedin a given assay.

In another embodiment, the control comprises contacting a controlpopulation of insulin secreting cells with an amount of ApoCIIIeffective to increase density and/or conductivity of Ca_(v)1 channels,in the presence of the one or more test compounds, and furthercontacting the control population of insulin secreting cells with a Srckinase inhibitor and/or a PKA inhibitor, wherein positive test compoundsthat inhibit the ApoCIII-induced increase in density and/or conductivityof Ca_(v)1 channels in the first population of insulin secreting cellsto a greater degree than in the Src kinase inhibitor and/or a PKAinhibitor control population of insulin secreting cells are candidatecompounds for limiting development of and/or treating diabetes.Exemplary Src kinase inhibitors include PP1 analogs, PP2, and compoundsdisclosed in the examples that follow. Exemplary PKA inhibitors includeadenosine 3′,5′-cyclic monophosphorothioate-R, H-7, H-8, H-9, H-89, andcompounds disclosed in the examples that follow.

As shown in the examples that follow, the inventors have discovered thatApoCIII hyperactivates β cell Ca_(v)1 channels throughintegrin-dependent co-activation of PKA and Src kinase. Thus, inhibitorsof PKA and/or Src should down-regulate positive candidate compounds ofthe present invention. Any suitable PKA and/or Src kinase inhibitor canbe used, including but not limited to those disclosed in the examplesthat follow. It is within the level of those of skill in the art todetermine, based on the teachings herein, the amount of any Src kinaseinhibitor(s) and/or a PKA inhibitor(s) that can be usefully used in agiven assay.

In a further embodiment, the control comprises contacting a controlpopulation of insulin secreting cells with an amount of ApoCIIIeffective to increase density and/or conductivity of Ca_(v)1 channels,in the presence of the one or more test compounds and further contactingthe control population of insulin secreting cells with a molecule thatinhibits β1 integrin expression or activity, wherein positive testcompounds that inhibit the ApoCIII-induced increase in density and/orconductivity of Ca_(v)1 channels in the first population of insulinsecreting cells to a greater degree than in the β1 integrin inhibitorcontrol population of insulin secreting cells are candidate compoundsfor limiting development of and/or treating diabetes. As shown in theexamples that follow, the inventors have discovered that ApoCIIIhyperactivates β cell Ca_(v)1 channels through β1 integrin-dependentcoactivation of PKA and Src kinase. Thus, inhibitors of β1 integrinshould down-regulate positive candidate compounds of the presentinvention. Any suitable β1 integrin inhibitor can be used (antibodies,antisense, siRNA, shRNA, etc.), including but not limited to thosedisclosed in the examples that follow. It is within the level of thoseof skill in the art to determine, based on the teachings herein, theamount of any β1 integrin inhibitor(s) that can be usefully used in agiven assay.

As will be understood by those of skill in the art, a single control canbe used in carrying out the methods of the invention, including but notlimited to any of the controls disclosed above. Alternatively, multiplecontrol embodiments can be used (2, 3, or more, including but notlimited to any of the controls disclosed above), wherein each embodimentutilizes a different control cell population.

In a second aspect, the present invention provides methods foridentifying candidate compounds for limiting development of and/ortreating diabetes, comprising

-   -   (a) contacting a first population of insulin secreting cells        with an amount of ApoCIII effective to increase density and/or        conductivity of Ca_(v)1 channels, in the presence of one or more        test compounds; and    -   (b) identifying those positive test compounds that inhibit β1        integrin expression or activity in the first population of        insulin secreting cells compared to control, wherein the        positive test compounds are candidate compounds for limiting        development of and/or treating diabetes.

In one embodiment, the control comprises contacting a second populationof insulin secreting cells contacted with an amount of ApoCIII effectiveto increase density and/or conductivity of Ca_(v)1 channels, in theabsence of one or more test compounds. This embodiment may comprise, forexample, contacting the second population of cells with a formulation,such as a buffer, that is similar to or identical to the formulation thetest compounds are dissolved in.

All embodiments of the first aspect of the invention can be used in thissecond aspect unless the context clearly dictates otherwise.

In one embodiment, the control comprises contacting a second populationof insulin secreting cells contacted with ApoCIII in the absence of testcompounds. This embodiment may comprise, for example, contacting thesecond population of cells with a formulation, such as a buffer, that issimilar to or identical to the formulation the test compounds aredissolved in.

In a third aspect, the present invention provides methods foridentifying candidate compounds for limiting development of and/ortreating diabetes, comprising

-   -   (a) contacting a first population of insulin secreting cells        with an amount of ApoCIII effective to increase density and/or        conductivity of Ca_(v)1 channels, in the presence of one or more        test compounds; and    -   (b) identifying those positive test compounds that inhibit        activation of PKA and/or Src kinase in the first population of        insulin secreting cells compared to control,

wherein the positive test compounds are candidate compounds for limitingdevelopment of and/or treating diabetes.

In one embodiment, the methods comprise identifying inhibitors of β1integrin-mediated activation of PKA and/or Src kinase.

In one embodiment, the control comprises contacting a second populationof insulin secreting cells contacted with an amount of ApoCIII effectiveto increase density and/or conductivity of Ca_(v)1 channels, in theabsence of one or more test compounds. This embodiment may comprise, forexample, contacting the second population of cells with a formulation,such as a buffer, that is similar to or identical to the formulation thetest compounds are dissolved in.

All embodiments of the first aspect of the invention can be used in thisthird aspect unless the context clearly dictates otherwise.

In a further aspect, the present invention provides methods for treatingor limiting development of diabetes, comprising administering to asubject in need thereof an amount effective of an inhibitor of PKA andSrc kinase to treat or limit development of diabetes. Exemplary Srckinase inhibitors include PP1 analogs, PP2, and compounds disclosed inthe examples that follow. Exemplary PKA inhibitors include adenosine3′,5′-cyclic monophosphorothioate-R, H-7, H-8, H-9, H-89, and compoundsdisclosed in the examples that follow.

In another aspect, the present invention provides methods for treatingor limiting development of diabetes, comprising administering to asubject in need thereof with an amount effective of an inhibitor of β1integrin expression and/or activity. In various embodiments, theinhibitor is selected from the group consisting of an anti-β1 integrinantibody, anti-β1 integrin aptamer, β1 integrin siRNA, β1 integrinshRNA, and β1 integrin antisense oligonucleotides.

In a still further aspect, the present invention provides methods fortreating or limiting development of diabetes, comprising administeringto a subject in need thereof with an amount effective of an inhibitor ofApoCIII activation of pancreatic β cells.

As used herein, an “inhibitor” of apoCIII activation includes compoundsthat reduce the transcription of apoCIII DNA into RNA, compounds thatreduce translation of the apoCIII RNA into protein, and compounds thatreduce the function of apoCIII protein. Such inhibiting can be completeinhibition or partial inhibition, such that the expression and/oractivity of the apoCIII is reduced, resulting in a reduced ability toincrease intracellular calcium concentration. Such inhibitors areselected from the group consisting of antibodies that bind to apoCIII;aptamers that can interfere with apoCIII activity; antisenseoligonucleotides directed against the apoCIII protein, DNA, or mRNA;small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) directedagainst the apoCIII protein, DNA, or mRNA, and any other chemical orbiological compound that can interfere with apoCIII activity.

In one embodiment of each of these therapeutic aspects, the method isfor treating diabetes. In this embodiment, the subject has beendiagnosed with type 1 or type 2 diabetes. As used herein, “diabetes” ischaracterized by insufficient or no production of insulin by thepancreas, leading to high blood sugar levels.

As used herein, “treating diabetes” means accomplishing one or more ofthe following: (a) reducing the severity of the diabetes or diabeticcomplications; (b) limiting or preventing development of diabeticcomplications; (c) inhibiting worsening of diabetic complications or ofsymptoms characteristic of diabetes; (d) limiting or preventingrecurrence diabetic complications or of symptoms characteristic ofdiabetes; (e) limiting or preventing recurrence of diabeticcomplications or of symptoms characteristic of diabetes in patients thatwere previously symptomatic.

Symptoms characteristic of diabetes include, but are not limited to,elevated blood glucose levels, decreased insulin production, insulinresistance, proteinuria, and impaired glomerular clearance. Diabeticcomplications that can be treated according to the methods of theinvention include, but are not limited to, complications in the nerves(such as diabetic neuropathy) and complications associated with smoothmuscle cell dysregulaton (including but not limited to erectiledysfunction, bladder dysfunction, and vascular complications includingbut not limited to atherosclerosis, stroke, and peripheral vasculardisease)

In another embodiment, the method is for limiting development ofdiabetes. In this aspect, the subject is at risk of type 1 or type 2diabetes, and a benefit is to limit development of diabetes and/ordiabetic complications. Any subject at risk of developing diabetes canbe treated, including but not limited to subjects with one or more ofmetabolic syndrome, known genetic risk factors for diabetes, a familyhistory of diabetes, and obesity.

In a further embodiment, the methods for treating or limitingdevelopment of diabetes and/or diabetic complications further comprisestreating those individuals that have been identified as overexpressingapoCIII compared to control. Increases in apoCIII expression precededevelopment of diabetic complications, and thus this embodiment permitsearly detection of suitable patients for treatment using the methods ofthe invention.

As used herein, “overexpression” is any amount of apoCIII expressionabove control. Any suitable control can be used, including apoCIIIexpression levels from a subject known not to be suffering fromdiabetes, or previously determined standardized expression levels ofapoCIII from a population of similar patient samples. Any amount ofincreased apoCIII expression relative to control is considered“overexpression”; in various embodiments, the overexpression comprisesat least 10%, 20%, 50%, 100%, 200%, or greater increased apoCIIIexpression compared to control. In a preferred embodiment, apoCIIIexpression is detected in blood or serum samples. In one embodiment toevaluate the levels of apoCIII in pos, neg, and control sera, albumin isremoved from serum samples using standard techniques, such as via use ofMontage Albumin Deplete Kit (Millipore) or AlbuSorb™ (Biotech SupportGroup). The collected sera samples can then be freeze-dried overnightand run on sep-Pak C18. The eluted proteins can be freeze-dried andthereafter dissolved in 100 μL, 0.1% TFA and run on an ACE C1810-×0.21-cm column 20-60%, and the area under the curve, where apoCIIIelutes, evaluated. ApoCIII can be identified using any suitabletechnique, including but not limited to MALDI mass spectrometry.

As used herein, the term “subject” or “patient” is meant any subject forwhich therapy is desired, including humans, cattle, dogs, cats, guineapigs, rabbits, rats, mice, insects, horses, chickens, and so on. Mostpreferably, the subject is human.

The therapeutic may be administered by any suitable route, including butnot limited to oral, topical, parenteral, intranasal, pulmonary, orrectal in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants and vehicles. The termparenteral as used herein includes percutaneous, subcutaneous,intravascular (e.g., intravenous), intramuscular, or intrathecalinjection or infusion techniques and the like. In addition, there isprovided a pharmaceutical formulation comprising a compound of theinvention and a pharmaceutically acceptable carrier The therapeutic maybe present in association with one or more non-toxic pharmaceuticallyacceptable carriers and/or diluents and/or adjuvants, and if desiredother active ingredients. The therapeutic may be in a form suitable fororal use, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, or syrups or elixirs.

The dosage range depends on the choice of the compound, the route ofadministration, the nature of the formulation, the nature of thesubject's condition, and the judgment of the attending practitioner. Forexample, oral administration would be expected to require higher dosagesthan administration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art

Example 1. Apolipoprotein CIII Hyperactivates β Cell Ca_(v)1 ChannelsThrough β1 Integrin-Dependent Coactivation of PKA and Sre Kinase

Summary

Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor oftriglyceride hydrolysis, but also participates in diabetes-relatedpathological events such as the inflammatory process and hyperactivationof voltage-gated Ca²⁺ (Ca_(v)) channels in the pancreatic β cell.However, nothing is known about the molecular mechanisms whereby ApoCIIIhyperactivates β cell Ca_(v) channels. We now demonstrate that ApoCIIIincreased Ca_(v)1 channel open probability and density. ApoCIII enhancedwhole-cell Ca²⁺ currents and the Ca_(v)1 channel blocker nimodipinecompletely abrogated this enhancement. The effect of ApoCIII was notsignificantly influenced by individual inhibition of PKA, PKC or Srckinase. However, combined inhibition of PKA, PKC and Src kinasecounteracted the effect of ApoCIII, similar results obtained bycoinhibition of PKA and Src kinase. Moreover, knockdown of β1 integrinprevented ApoCIII from hyperactivating β cell Ca_(v) channels. Thesedata reveal that ApoCIII hyperactivates β cell Ca_(v)1 channels throughβ1 integrin-dependent coactivation of PKA and Src kinase.

Introduction

Voltage-gated calcium (Ca_(v)) channels are critical in pancreatic βcell physiology and pathophysiology (Yang and Berggren, 2005; Yang andBerggren, 2006). They not only take center stage in the regulation ofinsulin secretion, but are also involved in β cell development, survivaland growth through the regulation of protein phosphorylation, geneexpression and the cell cycle (Yang and Berggren, 2005; Yang andBerggren, 2006). The function and density of β cell Ca_(v) channels areregulated by a wide range of mechanisms either shared by other celltypes or specific to β cells, e.g., channel phosphorylation, interactionwith other molecules and glucose metabolism-derived signaling(Catterall, 2000; Yang and Berggren, 2005; Yang and Berggren, 2006).Dysfunctional Ca_(v) channels cause β cell malfunction and even death asmanifested in the most common metabolic disorder diabetes mellitus (Yangand Berggren, 2005; Yang and Berggren, 2006). Indeed, aT-lymphocyte-mediated autoimmune attack plays a crucial role in β celldeath in type 1 diabetes. In addition, factors in type 1 diabetic serumcompel unphysiological amounts of Ca²⁺ to enter pancreatic β cellsthrough hyperactivation of β cell Ca_(v) channels resulting in β cellapoptosis. Undoubtedly, this process aggravates the disease developmenton top of the autoimmune attack (Yang and Berggren, 2005; Yang andBerggren, 2006). Such factors are also visualized in type 2 diabeticserum where they behave in the same way as they do in type 1 diabeticserum (Juntti-Berggren et al., 1993; Juntti-Berggren et al., 2004; Solet al., 2009). In fact, reduction in β cell mass and hyperactivation ofβ cell Ca_(v) channels appear under the type 2 diabetic conditions suchas those in the Goto-Kakizaki rat (Kato et al., 1996).

It has been demonstrated that elevated apolipoprotein CIII (ApoCIII)acts as a diabetologenic serum factor to drive β cell destruction viahyperactivation of β cell Ca_(v) channels (Juntti-Berggren et al., 2004;Sol et al., 2009). Moreover, we have recently shown that in vivosuppression of ApoCIII delays onset of diabetes in the BioBreeding rat,a rat model for human type 1 diabetes (Holmberg et al., 2011). Normally,ApoCIII is a blood plasma component. It is synthesized predominantly inthe liver and to a minor extent in the intestine. Liver and intestinalcells release this apolipoprotein into the blood where it is situated onthe surface of chylomicrons, very low density lipoproteins (LDLs) andhigh density lipoproteins (HDLs) (Gangabadage et al., 2008; Jong et al.,1999). ApoCIII is composed of 79 amino acid residues that form sixamphiphilic α-helixes, each containing about 10 residues. Thethree-dimensional NMR structure and dynamics of ApoCIII have beenresolved when it complexes with sodium dodecyl sulfate micelles,mimicking its natural lipid-bound state. The six amphiphilic α-helixesassemble into a necklace-like chain wrapping around the sodium dodecylsulfate micelle surface (Gangabadage et al., 2008). Dogmatically,ApoCIII serves as an effective inhibitor of triglyceride hydrolysis byinhibiting lipoprotein lipase and through interference withtriglyceride-rich lipoproteins binding to the negatively charged cellsurface where lipoprotein lipases and lipoprotein receptors reside(Gangabadage et al., 2008; Jong et al., 1999). It impedes the selectiveuptake of cholesteryl esters from LDL and HDL by binding to thescavenger receptor class B type I (SR-BI), and hampers the endocytosisof cholesterol-rich LDL by prevention of apolipoprotein B binding to LDLreceptors (Clavey et al., 1995; Huard et al., 2005; Xu et al., 1997).Elevated plasma ApoCIII concentration is a feature of dyslipidemia inobesity and observed in both type 1 and type 2 diabetes (Chan et al.,2002; Juntti-Berggren et al., 2004; Sundsten et al., 2008), whereas agroup of Ashkennazi Jewish with reduced plasma ApoCIII concentrationmaintains cardiovascular health and greater insulin sensitivity with ageand reaches exceptional longevity (Atzmon et al., 2006).

In addition to the dogmatic roles in lipid metabolism, ApoCIII is also amultifaceted player in cell signaling. It can bind to distinct cellsurface receptors including scavenger receptor class B type I (SR-BI),Toll-like receptor 2 (TLR2) and uncharacterized binding sites relayingcorresponding signals to their downstream effectors, e.g., β1 integrin,pertussis toxin-sensitive G proteins, NF-κB and protein kinases (Fangand Liu, 2000; Kawakami et al., 2006; Kawakami et al., 2007; Xu et al.,1997). However, nothing is known about the molecular mechanisms wherebyApoCIII hyperactivates β cell Ca_(v) channels. In the present study, wedemonstrate that ApoCIII upregulates β cell Ca_(v)1 channels through β1integrin-dependent coactivation of PKA and Src kinase.

Results

Apolipoprotein CIII Increases Ca_(v)1 Channel Density and Conductivityin the β Cell

Our previous work reveals that ApoCIII incubation significantly enhanceswhole-cell Ca²⁺ currents in the mouse islet β cell (Juntti-Berggren etal., 2004). To clarify what type of β cell Ca_(v) channels and whetherthe density or conductivity was affected, we analyzed unitary Ca_(v)1channel currents, characterized by a large unitary Ba²⁺ conductance withlong-lasting openings, in mouse islet β cells (FIG. 1A) and RINm5F cells(FIG. 1C) following ApoCIII incubation. In experiments with mouse isletβ cells, we observed more Ca_(v)1 channels, reflected by more layers ofunitary Ba²⁺ currents, in plasma membrane patches of ApoCIII-treatedcells than in those of control cells (FIG. 1A). The average number, openprobability and mean open time of unitary Ca_(v)1 channels inApoCIII-treated cells (n=32) were significantly greater than those incells exposed to control vehicle (n=33) (FIG. 1B). The mean closed timeof unitary Ca_(v)1 channels recorded in patches of ApoCIII-incubatedcells was significantly shorter than that in control patches (FIG. 1B).Likewise, similar effects of ApoCIII occurred on Ca_(v)1 channels ininsulin-secreting RINm5F cells. Plasma membrane patches ofApoCIII-incubated cells accommodated more Ca_(v)1 channels in comparisonwith those of vehicle-treated cells (FIG. 1C). Ca_(v)1 channels in theformer opened more frequently than those in the latter (FIG. 1C).ApoCIII incubation (n=35) significantly increased channel number,elevated open probability, prolonged mean open time and shortened meanclosed time of Ca_(v)1 channels as compared with incubation with vehiclesolution (n=34) (FIG. 1D). Obviously, the data reveal that ApoCIIIincreased both density and conductivity of β cell Ca_(v)1 channels.

Pharmacological Ablation of Ca_(v)1 Channels Prevents ApolipoproteinCIII-Induced Hyperactivation of β Cell Ca_(v) Channels

The verification of the effects of ApoCIII on Ca_(v)1 channels by singlechannel analysis does not necessarily mean that ApoCIII only attacksCa_(v)1 channels. To examine if the effects also occur on other types ofCa_(v) channels, we analyzed whole-cell Ca²⁺ currents in RINm5F cellsfollowing ApoCIII incubation in the absence and presence of the Ca_(v)1channel blocker nimodipine. Whole-cell Ca²⁺ currents in cells incubatedwith ApoCIII were larger than those in cells treated with vehiclesolution (FIG. 2A). Whole-cell Ca²⁺ current densities observed in thevoltage range from 10 to 30 mV in the ApoCIII group were significantlyhigher than those in the control group (FIG. 2B). In striking contrast,whole-cell Ca²⁺ currents were similar between control cells and cellsincubated with ApoCIII in the presence of nimodipine (FIG. 2C). Therewas no significant difference in the whole-cell Cu²⁺ current densitybetween the two treatments (FIG. 2D). The data confirm that ApoCIIIsolely impinge on β cell Ca_(v)1 channels.

Apolipoprotein CIII Hyperactivates Cell Ca_(v) Channels Via Coactivationof PKA and Sre Kinase

The increase in open probability of β cell Ca_(v)1 channels by ApoCIIIand the mediating role of protein kinases in ApoCIII signaling suggestthat ApoCIII may signal upstream of some protein kinases tohyperactivate β cell Ca_(v) channels (Gui et al., 2006; Kawakami et al.,2006; Rueckschloss and Isenberg, 2004; Waitkus-Edwards et al., 2002; Wuet al., 2001). Therefore, we explored the involvement of PKA, PKC andSrc kinase in ApoCIII-induced hyperactivation of β cell Ca_(v) channels.

First, we examined the effect of the PKA inhibitor H-89 onApoCIII-induced hyperactivation of β cell Ca_(v) channels in RINm5Fcells. Whole-cell Ca²⁺ currents registered in control cells were largerthan those in cells treated with ApoCIII, whereas whole-cell Ca²⁺currents recorded in cells incubated with ApoCIII plus H-89 sized inbetween (FIG. 3A). Average Ca²⁺ current densities measured inApoCIII-treated cells (filled circles, n=36) were significantly higherthan those in vehicle control cells (open circles, n=37) at voltagesranging from 10 to 50 mV (FIG. 3B). However, cells following cotreatmentof ApoCIII and H-89 (filled triangles, n=36) did not significantlydiffer from either cells treated with ApoCIII or control cells in termsof Ca²⁺ current density (FIG. 3B). Moreover, H-89 treatment did notsignificantly influence Ca²⁺ current densities under basal conditions,i.e. in the absence of ApoCIII (FIGS. 7A and B). The results indicatethat PKA inhibition marginally reduced ApoCIII-induced hyperactivationof β cell Ca_(v) channels.

Second, we tested the effect of the PKC inhibitor calphostin C (CalpC)on ApoCIII-induced hyperactivation of β cell Ca_(v) channels in RINm5Fcells. We observed that cells incubated with ApoCIII andApoCIII/CalpC-cotreated cells displayed similar whole-cell Ca²⁺currents, which were larger than those acquired in vehicle-treated cells(FIG. 3C). Mean Ca²⁺ current densities in ApoCIII-treated cells (filledcircles, n=33) at the voltage range 10-50 mV and cells exposed toApoCIII/CalpC (filled triangles, n=33) at a voltage range from 20 to 50mV increased significantly in comparison with vehicle control cells(open circles, n=33) (FIG. 3D). There is no difference betweenApoCIII-treated cells and ApoCIII/CalpC-cotreated cells with regard tothe Ca²⁺ current density (FIG. 3D). Furthermore, cells exposed tocontrol vehicle were similar to a CalpC-treated cells in terms of Ca²⁺current density (FIGS. 7C and D). The data demonstrate that PKCinhibition does not affect ApoCIII-induced hyperactivation of β cellCa_(v) channels.

Third, we evaluated the effect of the Src kinase inhibitor PP2 onApoCIII-induced hyperactivation of β cell Ca_(v) channels in RINm5Fcells. We found smaller and larger whole-cell Ca²⁺ currents in cellsfollowing incubation with vehicle solution and ApoCIII-incubated cells,respectively (FIG. 3E). Cells exposed to ApoCIII and PP2 fell betweenvehicle control cells and cells treated with ApoCIII with regard towhole-cell Ca²⁺ currents (FIG. 3E). Whole-cell Ca²⁺ current densitiesquantified in cells treated with ApoCIII (filled circles, n=40) at thevoltage range 10-50 mV were significantly elevated as compared withthose determined in vehicle control cells (open circles, n=40) (FIG.3F). Cells subjected to cotreatment of ApoCIII and PP2 (filledtriangles, n=40) showed significantly larger Ca²⁺ currents at thevoltage range 20-40 mV than vehicle control cells (open circles, n=40).However, the difference in the Ca²⁺ current density betweenApoCIII/PP2-cotreated cells and cells incubated with vehicle solution isless prominent than that between cells treated with ApoCIII and vehiclecontrol cells (FIG. 3F). Moreover, vehicle-treated cells (open circles,n=20) and cells incubated with PP2 (filled circles, n=19) exhibitedsimilar Ca²⁺ current densities (FIGS. 7E and F). The results suggestthat Src kinase inhibition has a tendency to decrease ApoCIII-inducedhyperactivation of β cell Ca_(v) channels.

The marginal and null effects of PKA, PKC or Src kinase inhibitors onApoCIII-induced hyperactivation of β cell Ca_(v) channels made us wonderwhat happens if a more complex inhibition of all these kinases isapplied. To address this question, we characterized the effect of theprotein kinase inhibitor cocktail H-89, CalpC and PP2 on ApoCIII-inducedhyperactivation of β cell Ca_(v) channels in RINm5F cells. Largerwhole-cell Ca²⁺ currents appeared in an ApoCIII-treated cells, whereassmaller whole-cell Ca²⁺ currents occurred in vehicle control cells andcells treated with ApoCIII in the presence of H-89, CalpC and PP2 (FIG.4A). ApoCIII treatment (filled circles, n=35) significantly increasedCa²⁺ current densities at the voltage range 10-50 mV as compared withvehicle control (open circles, n=35) and treatment with ApoCIII togetherwith H-89, CalpC and PP2 (filled triangles, n=34). The profile of Ca²⁺current densities in cells exposed to ApoCIII in the presence of H-89,CalpC and PP2 resembled that in vehicle control cells (FIG. 4B).Furthermore, treatment of control cells with the protein kinaseinhibitor cocktail H-89, CalpC and PP2 had no significant effect onwhole-cell Ca²⁺ currents under basal conditions, i.e. in the absence ofApoCIII (FIGS. 8A and B). The results demonstrate that combinedinhibition of PKA, PKC and Src kinase effectively ablatesApoCIII-induced hyperactivation of β cell Ca_(v) channels.

The marginal effect of PKA or Src kinase inhibitors alone on whole-cellCa²⁺ currents inevitably raised the question if coinhibition of PKA andSrc kinase is sufficient to prevent ApoCIII-induced hyperactivation of βcell Ca_(v) channels. We answered the question by analyzing whole-cellCa²⁺ currents in RINm5F cells following cotreatment of H-89 and PP2. Weobserved that whole-cell Ca²⁺ currents in ApoCIII-treated cells werelarger than those in control cells or cells subjected to treatment ofApoCIII in the presence of H-89 and PP2 (FIG. 4C). Significantly higherdensities of whole-cell Ca²⁺ currents appeared in the ApoCIII group(filled circles, n=26) in comparison with control group (open circles,n=26) or group subjected to incubation with ApoCIII in the presence ofH-89 and PP2 (filled triangles, n=27) (FIG. 4D). Moreover, whole-cellCa²⁺ currents in control cells resembled those observed in cells treatedwith H-89 and PP2 (FIGS. 8C and D). The data reveal that ApoCIIIenhances whole-cell Ca²⁺ currents via coactivation of PKA and SrcKinase.

Apolipoprotein CIII does not Influence β Cell Ca_(v)1 Channel Expression

Overnight incubation with ApoCIII may influence β cell Ca_(v)1 channelexpression. To test for this possibility, we analyzed β cell Ca_(v)1channel expression in RINm5F cells following ApoCIII incubation. Wefound that anti-Ca_(v)1.2, anti-Ca_(v)1.3 and anti-GAPDH antibodiesdetected clear Ca_(v)1.2, Ca_(v)1.3 and GAPDH immunoreactive bands,respectively. Control and ApoCIII-treated samples gave similarintensities of Ca_(v)1.2, Ca_(v)1.3 and GAPDH immunoreactivities (FIG.5A). FIG. 5B shows that there was no significant difference in therelative abundance of Ca_(v)1.2 (hatched column, n=6) and Ca_(v)1.3subunits (filled column, n=6) in RINm5F cell homogenates subjected toApoCIII incubation in comparison with vehicle incubation (open column,n=6) (P>0.05). The data reveal that ApoCIII incubation did not alter βcell Ca_(v)1 channel expression at the protein level.

Apolipoprotein Upregulates β Cell Ca_(v) Channels Via β1 Integrin

β1 integrin has been verified to serve as a mediator between ApoCIII anda certain number of protein kinases including PKA and Sic kinase (Gui etal., 2006; Kawakami et al., 2006; Rueckschloss and Isenberg, 2004;Waitkus-Edwards et al., 2002; Wu et al., 2001). This together with ourresults that ApoCIII hyperactivated β cell Ca_(v) channels viacoactivation of PKA and Src kinase raises the possibility for β1integrin to mediate ApoCIII-induced hyperactivation of β cell Ca_(v)channels. We investigated this possibility by implementing RNAinterference in combination with whole-cell Ca²⁺ analysis in RINm5Fcells. It turned out that transfection with two β1 integrin siRNAssignificantly decreased β1 integrin expression at the protein level(FIGS. 6A and B). We also visualized that whole-cell Ca²⁺ currentsdetected in cells subjected to negative control siRNA transfection andApoCIII exposure (NC siRNA/apoCIII) were larger than those in othercells following different treatments (FIG. 6C). These treatmentsincluded mock transfection and control vehicle incubation (NOsiRNA/Control), negative control siRNA transfection and exposure tocontrol vehicle (NC siRNA/Control), β1 integrin siRNA transfection andtreatment with vehicle solution (β1 integrin siRNA/Control), and β1integrin siRNA transfection and ApoCIII incubation (β1 integrinsiRNA/ApoCIII). Cells subsequent to NC siRNA/apoCIII (filled triangles,n=28) showed a significant increase in Ca²⁺ current density at thevoltage range 10-40 mV in comparison with NO siRNA/Control (n=29), NCsiRNA/Control (n=28), β1 integrin siRNA/Control (n=29), and β1 integrinsiRNA/ApoCIII (n=29) (FIG. 6D). The difference in Ca²⁺ current densitybetween NC siRNA/apoCIII and β1 integrin siRNA/apoCIII was less thanthat between NC siRNA/apoCIII and other treatments (FIG. 6D). Takentogether, the results demonstrate that ApoCIII critically relies on β1integrin to hyperactivate β cell Ca_(v) channels.

Discussion

The gross conductivity of Ca _(V) channels depends on the density andactivity of functional channels in the plasma membrane of the cell.Enhancement of whole-cell Ca²⁺ currents by type 1 diabetic serum and itsfactor ApoCIII can result from enriched density and/or increasedconductivity of functional Ca_(v) channels in the β cell plasma membrane(Juntti-Berggren et al., 1993; Juntti-Berggren et al., 2004). However,all studies (Juntti-Berggren et al., 2004; Ristic et al., 1998; Yang andBerggren, 2005; Yang and Berggren, 2006) except one (Juntti-Berggren etal., 1993) have so far examined the effect of type 1 diabetic serum onCa_(v) channels only at the whole cell level. In the study byJuntti-Berggren et al, the increase in β cell Ca_(v) channel activity bytype 1 diabetic serum was characterized at both the single channel andthe whole-cell level (Juntti-Berggren et al., 1993). Unfortunately, thiswork did not analyze whether type 1 diabetic serum could alter thedensity of functional Ca_(v) channels in the β cell plasma membrane(Juntti-Berggren et al., 1993). Although we have previously revealedthat ApoCIII serves as a type 1 diabetic serum factor, hyperactivating βcell Ca_(v) channels, only whole-cell patch-clamp analysis was performed(Juntti-Berggren et al., 2004). Undoubtedly, detailed examination ofbiophysical properties of single Ca_(v) channels in ApoCIII-treatedcells should be implemented to mechanistically dissect hyperactivationof β cell Ca_(v) channels by this apolipoprotein. Interestingly,cell-attached single channel recordings in the present work reveals thatincubation with ApoCIII not only augments the activity of individual βcell Ca_(v)1 channels but also enriches the number of functional Ca_(v)1channels in the recorded area of the β cell plasma membrane. Theaugmentation of single Ca_(v)1 channel activity is visualized as anincreased open probability attributed to the prolonged mean open timeand shortened mean closed time. Enrichment of number of functionalCa_(v)1 channels is verified by appearance of more levels of singleCa_(v)1 channel conductance.

The insulin-secreting RINm5F cell is equipped with Ca_(v)1, Ca_(v)2 andCa_(v)3 channels (Yang and Berggren, 2005; Yang and Berggren, 2006). Weinvestigated if ApoCIII selectively hyperactivates Ca_(v)1 channels orindiscriminately impacts all these three types of Ca_(v) channels inthis insulin-secreting cell. It turned out that ApoCIII-inducedhyperactivation of β cell Ca_(v) channels could no longer take placefollowing pharmacological ablation of Ca_(v)1 channels. This means thatApoCIII selectively hyperactivates Ca_(v)1 channels, which are the majorCa_(v) channel type playing a predominant role over other types ofCa_(v) channels in β cell physiology and pathophysiology. The selectivehyperactivation of β cell Ca_(v)1 channels by ApoCIII accounts for thepathophysiological role of this lipoprotein in Ca²⁺-dependent β celldeath (Juntti-Berggren et al., 2004; Yang and Berggren, 2005; Yang andBerggren, 2006).

A series of protein kinases, such as PKA and PKC, can effectivelyphosphorylate Ca_(v) channels resulting in increases in the open channeldensity and activity due to phosphorylation-induced conformationalchanges in these channels (Catterall, 2000; Kavalali et al., 1997; Yangand Tsien, 1993). Increases in the number and open probability offunctional Ca_(v) channels by ApoCIII might be mediated by proteinkinases. ApoCIII has been demonstrated to activate PKC through β1integrin in monocytic cells (Kawakami et al., 2006). Furthermore, β1integrin activation can also upregulate Ca_(v)1 channels in neurons,ventricular myocytes and vascular smooth muscle cells throughstimulation of PKA, PKC and Src kinase (Gui et al., 2006; Rueckschlossand Isenberg, 2004; Waitkus-Edwards et al., 2002; Wu et al., 2001). Allthese components are present in β cells (Bosco et al., 2000; Kantengwaet al., 1997; Mukai et al., 2011; Nikolova et al., 2006; Yang andBerggren, 2006) and may suggest that ApoCIII employs the β1integrin-PKA/PKC/Sre kinase cascade to hyperactivate β cell Ca_(v)channels. Indeed, the present work shows that complex inhibition of PKA,PKC and Src kinase effectively abrogates ApoCIII-induced hyperactivationof β cell Ca_(v) channels and that coinhibition of PKA and Src kinase isenough for this effect. However, individual inhibition of PKA, PKC orSrc kinase only produced, if anything, a marginal effect onApoCIII-induced hyperactivation of β cell Ca_(v) channels. Hence, weconclude that ApoCIII relies on parallel PKA and Src pathways toupregulate β cell Ca_(v) channels.

Occurrence of ApoCIII-induced hyperactivation of β cell Ca_(v) channelsrequires overnight incubation. Hence, the effect might be accounted forby an increase in Ca_(v) channel expression. Therefore, we quantifiedimmunoreactivities of Ca_(v)1.2 and Ca_(v)1.3 subunits in RINm5F cellsfollowing overnight incubation with ApoCIII. However, the incubation hadno influence on β cell Ca_(v)1 channel expression. We therefore excludedthe possibility that ApoCIII elevates β cell Ca_(v)1 channel expression.

The transmembrane receptor β1 integrin is noncovalently associated withother integrins to form a set of heterodimers. They recognize a largenumber of soluble and surface-bound proteins to mediate cell-cell,cell-extracellular matrix and cell-pathogen interactions (Luo et al.,2007). β1 Integrin is situated downstream of ApoCIII and upstream ofPKA/PKC/Src kinase in some cell types (Gui et al., 2006; Kawakami etal., 2006; Rueckschloss and Isenberg, 2004; Waitkus-Edwards et al.,2002; Wu et al., 2001). This made us investigate whether the ApoCIII-β1integrin-PKA/PKC/Src kinase pathway operates in the β cell as themechanism whereby this apolipoprotein hyperactivates Ca_(v)1 channels.Interestingly, knockdown of β1 integrin does not influence β cell Ca_(v)channel activity in the absence of ApoCIII, but significantly abrogatesApoCIII-induced hyperactivation of β cell Ca_(v) channels. The resultsclearly verify that β1 integrin plays a significant role in mediatingthe action of ApoCIII on β cell Ca_(v)1 channel activity.

In conclusion, our findings demonstrate that ApoCIII selectivelyhyperactivates β cell Ca_(v)1 channels through parallel PKA and Srckinase pathways in a β1 integrin-dependent fashion. ApoCIII-inducedhyperactivation of β cell Ca_(v)1 channels is characterized by theenriched density and increased activity of functional Ca_(v)1 channelsin the β cell plasma membrane. Undoubtedly, this novelsignal-transduction pathway has a potential to serve as an innovativedrug discovery platform for the prevention of Ca²⁺-dependent β celldeath in association with diabetes.

EXPERIMENTAL PROCEDURES

Cell Culture and Treatments

Islets of Langerhans were isolated from adult male and female mice anddispersed into single β cells. RINm5F cells at about 70% confluency weretrypsinized. The resultant suspension of cells was seeded into Petridishes or 12-well plates. The cells were cultivated in RPMI 1640 mediumsupplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 100U/100 μg/ml penicillin/streptomycin (Invitrogen, Carlsbad, Calif.) andmaintained at 37° C. in a humidified 5% CO₂ incubator. They were grownovernight and then subjected to siRNA transfection. For patch-clampanalysis, cells underwent overnight treatment with ApoCIII, the PKAinhibitor H-89 (Calbiochem, La Jolla, Calif.), the PKC inhibitorcalphostin C (Calbiochem), the Src kinase inhibitor PP2 (Calbiochem) andthe Ca_(v)1 channel blocker nimodipine (Calbiochem) in RPMI medium atfinal concentrations of 20 μg/ml, 0.5 μM, 0.1 μM, and 5 μM,respectively. ApoCIII was dissolved in 0.1% triflouroacetic acid (TFA)to make a stock solution of 1 mg/ml, whereas H-89, calphostin C, PP2 andnimodipine were dissolved in dimethyl sulfoxide (DMSO) to form stocksolutions of 5 mM, 1 mM and 10 mM, respectively. 0.002% TFA and/or 0.03%DMSO were used as vehicle controls.

siRNA Design and Transfection

Two pairs of 21-mer siRNA duplexes targeting the rat β1 integrin (β1integrin siRNA #1, ID127971 and β1 integrin siRNA #2, ID127972) weredesigned and chemically synthesized by Applied Biosystems/Ambion(Austin, Tex). Their sequences were subjected to BLAST search to ensuretheir specificity. Silencer® Select Negative Control siRNA. (4390843),not targeting any gene product, and Silencer® Select GAPDH PositiveControl siRNA (4390849), efficiently silencing GAPDH in human, mouse,and rat cells, were purchased from Applied Biosystems/Ambion (Austin,Tex.). RINm5F cells were reversely transfected with Lipofectamine™RNAiMAX™. Briefly, negative control siRNA, β1 integrin siRNA #1 or β1integrin siRNA #2 was mixed with Lipofectamine™ RNAiMAX™ followed by20-min incubation at room temperature. Subsequently, cells were added tothe siRNA/Lipofectamine™ RNAiMAX mixtures followed by gentle agitationand kept at 37° C. in a humidified 5% CO₂ incubator. After 72 h, thetransfected cells were grown to about 70% confluency and subjected toimmunoblot assay or different treatments.

SDS-PAGE and Immunoblot Analysis

RINm5F cells following different treatments were lysed in a lysis buffer(pH 7.5) consisting of 50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA,10% glycerol, 1% triton X-100, 1 mM PMSF and a protease inhibitorcocktail (Roche Diagnostics, Mannheim, Germany). The lysate wascentrifuged at 800×g for 10 min at 4° C. to remove cell debris andnuclei. The protein concentration of the resulting samples wasdetermined with Bio-Rad protein assay reagent (Bio-Rad, Hercules,Calif.). The samples were then denatured by heating at 96° C. for 3 minin SDS sample buffer and underwent sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and immunoblot analysis. Briefly, 50, 90or 180 μg proteins were separated in discontinuous gels consisting of a4% acrylamide stacking gel (pH 6.8) and an 8% acrylamide separating gel(pH 8.8). The separated proteins were then electroblotted to hydrophobicpolyvinylidene difluoride membrane (Hybond-P, GE Healthcare, Uppsala,Sweden). The blots were blocked by incubation for 1 h with 5% non-fatmilk powder in a washing buffer, containing 50 mMtris(hydroxymethyl)aminomethane, 150 mM NaCl and 0.05% Tween 20 (pH7.5). They were then incubated overnight at 4° C. with affinity-purifiedrabbit polyclonal antibodies to β1 integrin (1:500; Millipore,Billerica, Mass.), Ca_(v)1.2 (1:200) and Ca_(v)1.3 (1:200),respectively, and for 1 h at room temperature with mouse monoclonalantibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:4000;Applied Biosystems/Ambion, Austin, Tex.), respectively. After rinsingwith the washing buffer, the blots were incubated with the secondaryantibodies (either horseradish peroxidase-conjugated goat anti-rabbitIgG or horseradish peroxidase-conjugated goat anti-mouse IgG; 1:50,000;Bio-Rad, Hercules, Calif.) at room temperature for 45 min. Theimmunoreactive bands were visualized with the ECL plus Western blottingdetection system (GE Healthcare, Uppsala, Sweden).

Electrophysiology

Mouse islet cells and RINm5F cells following different treatments weresubjected to single channel and whole-cell patch-clamp measurements.Cell-attached and perforated whole-cell patch-clamp configurations wereemployed. Electrodes were made from borosilicate glass capillaries,fire-polished and coated with Sylgard close to their tips. Some of themwere filled with a solution containing (in mM) 110 BaCl₂, 10 TEA-Cl, and5 HEPES (pH 7.4 with Ba(OH)₂) for single channel measurements. Otherswere filled with a solution composed of (in mM) 76 Cs₂SO₄, 1 MgCl₂, 10KCl, 10 NaCl, and 5 HEPES (pH 7.35 with CsOH), as well as amphotericin B(0.24 mg/ml) for whole-cell current recordings. Electrode resistanceranged between 4 and 6 MΩ when they were filled with electrode solutionsand immersed in bath solutions. The electrode offset potential wascorrected in bath solutions prior to gigaseal formation. Single-channelrecordings were performed with cells bathed in a depolarizing externalrecording solution, containing (in nM) 125 KCl, 30 KOH, 10 EGTA, 2CaCl₂, 1 MgCl₂, and 5 HEPES-KOH (pH 7.15). This solution was used tobring the intracellular potential to 0 mV. For perforated whole-cellcurrent measurements, the cells were bathed in a solution containing (inmM) 138 NaCl, 5.6 KCl, 1.2 MgCl₂, 10 CaCl₂, 5 HEPES (pH 7.4). Singlechannel and whole-cell currents were recorded with an Axopatch 200Bamplifier (Molecular Devices, Foster City, Calif.) and an EPC-9 patchclamp amplifier (HEKA Elektronik, Lambrecht/Pfalz, Germany),respectively, at room temperature (about 22° C.). Acquisition andanalysis of single channel and whole-cell current data were done usingthe software program pCLAMP 10 (Axon Instruments) and the softwareprogram PatchMaster/FitMaster (HEKA), respectively. The amplitude ofwhole-cell currents was normalized by the cell capacitance.

Statistical Analysis

All data are presented as mean±SEM. Statistical significance wasdetermined by one-way ANOVA, followed by least significant difference(LSD) test. When two groups were compared, unpaired Student's t test orMann-Whitney U test was employed. The significance level was set at 0.05or 0.01.

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We claim:
 1. A method for treating or limiting development of diabetes,comprising administering to a subject in need thereof an effectiveamount of an inhibitor of a protein kinase A and an inhibitor of Srckinase to treat or limit development of diabetes.
 2. The method of claim1, wherein the PKA inhibitor is selected from the group consisting of3′,5′-cyclic monophosphorothioate-R, H-7, H-8, H-9, and H-89.
 3. Themethod of claim 1, wherein the Src kinase inhibitor is selected from thegroup consisting of PP1 and PP2.
 4. The method of claim 1, wherein themethod is for treating diabetes.
 5. The method of claim 1, wherein themethod is for limiting development of diabetes.
 6. The method of claim1, wherein the subject has or is at risk of developing type 1 diabetes.7. The method of claim 1, wherein the subject has or is at risk ofdeveloping type 2 diabetes.
 8. The method of claim 1, wherein thesubject has type 2 diabetes.
 9. The method of claim 1, wherein thesubject overexpresses apoCIII relative to control.
 10. The method ofclaim 1, wherein the method is for treating diabetes.
 11. The method ofclaim 2, wherein the method is for limiting development of diabetes. 12.The method of claim 2, wherein the subject has or is at risk ofdeveloping type 1 diabetes.
 13. The method of claim 2, wherein thesubject has or is at risk of developing type 2 diabetes.
 14. The methodof claim 2, wherein the subject has type 2 diabetes.
 15. The method ofclaim 2, wherein the subject overexpresses apoCIII relative to control.16. The method of claim 3, wherein the method is for treating diabetes.17. The method of claim 3, wherein the method is for limitingdevelopment of diabetes.
 18. The method of claim 3, wherein the subjecthas or is at risk of developing type 1 diabetes.
 19. The method of claim3, wherein the subject has or is at risk of developing type 2 diabetes.20. The method of claim 3, wherein the subject has type 2 diabetes. 21.The method of claim 3, wherein the subject overexpresses apoCIIIrelative to control.